Composition and method for controlling intestinal pathogenic organisms

ABSTRACT

An antigen composition for stimulating an immune response in an inoculated avian species to at least one intestinal pathogenic organism includes seven field strains of  E. coli, Pseudomona aeruginosa, Aerobacter aerogenes, Salmonella enteritidis, Salmonella typhimurium, Salmonella  agona, and  Salmonella  Kentucky. The antigen composition can be used alone or in combination with a Marek&#39;s Disease vaccine to reduce shedding of  E. coli  and/or  Salmonella  bacteria.

FIELD OF THE INVENTION

The invention pertains generally to composition for controllingintestinal pathogenic organisms in avian species and, more particularly,to a multivalent antigen for inducing immunity to specific bacterialdiseases and/or to enhance immunity in an infected organism.

BACKGROUND OF THE INVENTION

Consumption of poultry products contaminated with Salmonella bacteria isa significant source of gastrointestinal infections in humans. Forexample, Salmonella enteritidis, especially phage type 4, has becomemore common in both poultry and humans since the early 1980's. Theprevalence of Salmonella typhimurium, on the other hand, has remainedrelatively stable. However, the spread of the antibiotic-resistantstrain DT104 in domestic flocks gives some reason for concern.Accordingly, the presence of Salmonella in commercial meat and foodproducts is a major public health concern given that such infections canlead to serious illness or, in severe cases, death. Further, Salmonellainfections in chickens, turkeys and ducks raise concerns for poultryproducers due to increasing rates of morbidity and mortality as well aslosses attributable culling and/or rejection of infected birds.

Salmonella infections can be spread via intraspecies or horizontaltransmission, i.e., from animal to animal, and/or via interspecies orvertical transmission, i.e., from animal to humans. Generally,horizontal transmission of Salmonella bacteria is typically via exposureto environmental factors such as, for example, contaminated feces,bedding, nesting materials and/or other fomites. In contrast, verticaltransmission of Salmonella bacteria is typically via oral exposure tothe bacteria such by handling contaminated raw meats. Verticaltransmission can also occur via shell contamination and/or internaltransovarian contamination of the yolk of eggs produced by infectedbirds.

The basis for good control of Salmonella infections in farmenvironments, in particular, in poultry farms, is good farming andhygiene practices. Such practices include, for example, managing andpreventing contamination of feeds, monitoring of animal health, cleaningand disinfection of coops and pens, and control of pest species such as,for examples, rodents. Testing and removal of infected orpathogen-positive animals from production and/or contact with uninfectedanimals are also vital to controlling horizontal and/or verticaltransmission of such infections.

Poultry infected with Salmonella bacteria generally develop a strongimmune response to the pathogen which is typically manifested byprogressive reduction in excretion of the organism and reduced diseaseand excretion upon subsequent challenge. Accordingly, there is a needfor an effective means for inducing an immune response to Salmonellabacteria in poultry which results in reduced disease and excretion orshedding of the bacteria while reducing productivity losses attributableto culling and/or rejection of infected birds.

Recently, vaccination of commercial poultry flocks to increaseresistance against pathogenic exposure to Salmonella has become moreprevalent particularly in view of increasing public awareness. However,such vaccination programs are generally difficult, time consuming and/orprohibitively expensive to administer on a commercial production scale.Accordingly, there is a need for an effective means for vaccinatingdomestic poultry and fowl against Salmonella infections.

Additionally, it is generally believed that vaccination is not a controloption for serovars other than Salmonella enteritidis and Salmonellatyphimurium which can be present on poultry farms. It is also generallybelieved that vaccination has limited effect on improving animal healthand welfare and such vaccines are primarily used for public healthreasons. Accordingly, there is a need for an antigen composition orvaccine effective to result in improved avian health and welfare such ascan be manifested by increased weight gain and reduced mortality.

Further, some antigens may interfere with efficacy of other vaccines ormedications administered simultaneously with and/or subsequent tovaccination. Additionally or alternatively, particular antigens mayinterfere with or affect the accuracy of traditional test or screeningtools used to detect active or prior infection. Accordingly, there is ademand for a Salmonella antigen which can be administered to domesticpoultry and fowl which does not reduce the effectiveness of othervaccines such as, for example, Marek's disease vaccines.

SUMMARY OF THE INVENTION

A general object of the invention is to provide a multivalent antigenfor inducing an immune response and/or providing enhanced immunity to apathogenic organism such as Salmonella spp.

A more specific object of the invention is to overcome one or more ofthe problems described above.

The general object of the invention can be obtained, at least in part,through a multivalent antigen composition comprising seven field strainsof E. coli, Pseudomona aeruginosa, Aerobacter aerogenes, Salmonellaenteritidis, Salmonella typhimurium, Salmonella agona and SalmonellaKentucky. The composition induces an immune response in an inoculatedavian species to at least one intestinal pathogenic organism.

The prior art generally fails to provide a Salmonella-containingmultivalent antigen composition which is as effective as desired ininducing an immune response to at least one intestinal pathogenicorganism such as, for example, Salmonella spp. which is manifested by areduced fecal count in an inoculated avian species. The prior artfurther generally fails to provide a multivalent antigen compositionwhich can be easily and effectively administered in a commercial farmenvironment at a reduced cost. The prior art additionally fails toprovide a multivalent antigen composition that can be utilized alone orin combination with other vaccine products without reducing the efficacyof either vaccine component and/or the ability to detect or diagnoseparticular diseases within inoculated birds.

The invention further comprehends a bacterin vaccine comprising about67% of seven field strains of E. coli, about 10% Pseudomona aeruginosa,about 10% Aerobacter aerogenes, about 4% Salmonella enteritidis, about3% Salmonella typhimurium, about 3% Salmonella agona, and about 3%Salmonella Kentucky. The seven E. coli strains are selected from thegroup consisting of ATCC strain 25922, a University of Delaware fieldisolate, and five Delmarva field isolates.

The invention additionally comprehends an in ovo vaccine including abacterin vaccine and a Marek's disease vaccine. The bacterin vaccinecomprises seven field strains of E. coli, ATCC strain 27853 ofPseudomona aeruginosa, Aerobacter aerogenes, ATCC strain 13076 ofSalmonella enteritidis, ATCC strain 14028 of Salmonella typhimurium,Salmonella agona, and Salmonella Kentucky. At least one field strain ofE. coli consists of ATCC strain 25922 and each strain of E. coli ispresent in substantially equal amounts. The in ovo vaccine reduces aconcentration of at least one pathogenic organism in a gastrointestinaltract of an inoculated avian species.

As used herein the term “bacterin” or “bacterin vaccine” refers to avaccine composition generally comprised of dead or inactivated bacteriaspecies.

As used herein the terms “about” and “substantially” when used inconjunction with a percentage or the term “equal” refer to a valuefalling within a range of ±1 percentage point. For example, aconcentration of about 5% includes all concentrations falling within therange of 4% to 6%.

Other objects and advantages will be apparent to those skilled in theart from the following detailed description taken in conjunction withthe examples and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart showing the effect of an antigen composition of theinvention on Marek's Disease vaccine titration in culture.

FIG. 2 is a chart showing the effect of an antigen composition of theinvention on Marek's Disease vaccine re-isolation titers in vivo.

DETAILED DESCRIPTION

The invention provides a multivalent antigen or antigen compositionwhich stimulates an immune response in an inoculated avian species to atleast one intestinal pathogenic organism. The multivalent antigencomposition includes seven field strains of E. coli, Pseudomonaaeruginosa, Aerobacter aerogenes, Salmonella enteritidis, Salmonellatyphimurium, Salmonella agona and Salmonella Kentucky.

In accordance with certain embodiments, the multivalent antigen orantigen composition stimulates an immune response to an intestinalpathogenic organism selected from Clostridium perfringens, Salmonellaspp., E. coli or a combination thereof. Such immune response can bemanifested as a reduction in fecal bacterial counts for a particularpathogen such as, for example, reduction in Salmonella spp. fecalbacteria counts and/or E. coli fecal bacterial counts. Such immuneresponse can additionally or alternatively be manifested as a reductionin lesion formation upon exposure to Clostridium perfringens.

Various strains of E. coli bacteria can be included in the antigencomposition. Suitably, such strains of E. coli bacteria can be selectedfrom ATCC strain 25922, a University of Delaware field isolate, one ormore Delmarva field isolates or a combination thereof. In accordancewith one embodiment, the antigen composition includes seven fieldstrains of E. coli bacteria including ATCC strain 25922, a University ofDelaware field isolate and five Delmarva field isolates.

In accordance with certain embodiments, the antigen composition includesabout 67% of seven strains of E. coli bacteria. Suitably, each strain ofE. coli bacteria is present in an approximately equal amount.

Various strains of Pseudomona aeruginosa are suitable for use in theantigen composition. In accordance with one embodiment, the antigencomposition can include ATCC strain 27653 of Pseudomona aeruginosa.Suitably, the antigen composition can include about 10% Pseudomonaaeruginosa.

The antigen composition further includes Aerobacter aerogenes such as ina concentration of about 10%. In accordance with certain aspects of theinvention, the antigen composition is or should be free or devoid ofEnterobacter aerogenes and/or Klebsiella pneumoniae.

The antigen composition also includes at least four strains ofSalmonella species. In particular, the antigen composition includesSalmonella enteritidis, Salmonella typhimurium, Salmonella agona andSalmonella Kentucky. In accordance with certain embodiments, the antigencomposition can include ATCC strain 13076 of Salmonella enteritidisand/or ATCC strain 14028 of Salmonella typhimurium.

Suitably, the antigen composition, in accordance with one embodiment,can include about 4% Salmonella enteritidis, about 3% Salmonellatyphimurium, about 3% Salmonella agona and about 3% Salmonella Kentucky.

Suitably, the multivalent antigen or antigen composition can be utilizedas or in an in ovo vaccine for inoculating avian species or domesticfowl. For example, about 0.005 ml to about 0.05 ml of the multivalentantigen or antigen composition can be used to inoculate an embryonatedegg. In accordance with certain embodiments, the multivalent antigen orantigen composition can be given in a dose of about 0.0063 ml to about0.0375 ml per embryonated egg.

The multivalent antigen or antigen composition is suitable for use aloneas a bacterin vaccine or in combination with one or more other vaccinepreparations. For example, the antigen composition can be administeredsequentially with or simultaneously with another vaccine preparationsuch as, for example, a Marek's Disease vaccine.

In accordance with one embodiment, the antigen composition can be mixedor combined with a Marek's Disease vaccine. Such combined or mixedvaccine comprises a bacterin vaccine including seven strains of E. coli,Pseudomona aeruginosa, Aerobacter aerogenes, Salmonella enteritidis,Salmonella typhimurium, Salmonella agona and Salmonella Kentucky and aMarek's Disease vaccine. The Marek's Disease vaccine can include an HVTvaccine, a SB-1 vaccine or a bivalent vaccine including a mixture orcombination of HVT and SB-1 strains. Advantageously, the bacterin andMarek's Disease vaccine may be combined in any suitable ratio. Forexample, the combined vaccine may have a bacterin vaccine to Marek'sDisease vaccine ratio in the range of about 1:15 to 15:1. In accordancewith certain embodiments, the bacterin vaccine and the Marek's Diseasevaccine can be combined in a 1:1 ratio.

The combined bacterin-Marek's Disease vaccine reduces a concentration ofat least one pathogenic organism in a gastrointestinal tract of aninoculated avian species. Such pathogenic organism can include E. coli,Salmonella spp. or a combination thereof.

Suitably, the combined bacterin-Marek's Disease vaccine can be an in ovovaccine suitable for inoculating an avian species or domestic fowl suchas, for example, chickens, ducks, geese and/or turkeys. For example, thecombined bacterin-Marek's Disease vaccine can be administered in ovo ina dose of about 0.005 ml to about 0.1 ml combined vaccine perembryonated egg. In accordance with certain embodiments, a dose of thecombined bacterin-Marek's Disease vaccine can include about 0.0063 ml toabout 0.0375 ml bacterin vaccine.

A method for reducing transmission of pathogenic gastrointestinalorganisms includes inoculating an avian species in ovo at about 18 daysembryonic age with the above-described antigen composition alone suchas, for example, as a bacterin vaccine or in combination with anothervaccine preparation such as, for example, a Marek's Disease vaccine.

EXAMPLES Antigen Composition

An antigen composition was produced using various strains of bacteria,shown in TABLE 1, below, commonly found in poultry and/or humans. Eachbacteria isolate was initially individually grown in 1000 ml of NutrientBroth (Sigma N7519) at 35±1° C. for 24±2 hours. After the incubationperiod, each broth was centrifuged for approximately 10 minutes at 5000rpm in individual centrifuge sectors to separate the cells from thebroth. The supernatant was then aseptically removed from each centrifugevessel. The remaining cultures from each tube were then re-suspended inButterfield's Phosphate diluent and tested to determine purity. Thepurified cultures collectively formed a Master Seed.

The above steps were repeated until the quantity of Master Seed requiredto produce mass quantities of Working Seed stock was achieved. Followingdetermination of purity and specie, all Working Seed stock batches weremixed, separated into batch fermentation vessels and grown at 35±1° C.for 24±2 hour periods. At the completion of each batch, the entire batchwas carefully mixed and a sample of each culture was then plated ontoNutrient Agar. Colonies were counted after a further incubation periodof 24±2 hours at 35±1° C. using 10-fold dilutions up to 10¹⁰ dilutionrate. Plates with CFUs between 30 and 300 were counted.

TABLE 1 Bacterial component CFU Counts E. coli Isolate #1 1.36 × 10¹⁰ E.coli Isolate #2 2.03 × 10¹⁰ E. coli Isolate #3 6.80 × 10⁹ E. coliIsolate #4 2.92 × 10¹⁰ E. coli Isolate #5 1.28 × 10¹⁰ E. coli Isolate #62.13 × 10¹⁰ E. coli Isolate #7 5.30 × 10¹⁰ Pseudomona aeruginosa 2.14 ×10⁹ Aerobacter aerogenes 9.40 × 10⁸ Salmonella enteritidis 1.86 × 10⁹Salmonella typhimurium 2.38 × 10⁹ Salmonella agona 4.10 × 10⁹ SalmonellaKentucky 6.50 × 10⁹

The final counts were used to dilute and mix the individual culturesinto the final antigen composition or bacterin vaccine, as shown inTABLE 2, below. The bacteria were then killed by autoclaving at 121° C.for 15±2 minutes. This procedure was then repeated to ensure totalbacteria kill.

TABLE 2 Bacterial Component Concentration E. coli (7 field strains) 67%(each strain in ≈ equal amounts) Pseudomona Aeruginosa 10% Aerobacteraerogenes 10% Salmonella enteritidis 4% Salmonella typhimurium 3%Salmonella agona 3% Salmonella Kentucky 3%

Effect on Lesion Formation Due to Clostridium perfringens Exposure

Fourteen treatment groups of Ross (Male)×Cobb (Female) broilers were inovo inoculated with either a saline control or the antigen compositiondescribed in TABLE 2 at an embryonic age of 18 days. The fourteentreatment groups include seven (7) control groups each including 20 maleand 20 female chicks and seven (7) vaccine groups each including 20 maleand 20 female chicks.

All birds were inoculated with Clostridium perfringens (10⁴ per bird) onpost-hatch Day 8 to induce necrotic enteritis. Four male and four femalebirds from each control group and each vaccine group were humanelyeuthanized on Days 21 and 49, necropsied and the intestinal tractsvisually inspected for signs of necrotic enteritis and/or coccidiosis.The intestinal lesion scores including both coccidiosis signs andnecrotic enteritis signs were recorded. Lesions were scored on a scaleof 0 to 4 as follows:

-   -   0=No lesions found;    -   1=Slight redness with no cell sloughing (mucus);    -   2=Moderate redness and/or slight cell sloughing;    -   3=Severe redness and/or severe cell sloughing; and    -   4=Actual bleeding observed.

The lesion score data, as summarized in TABLE 3, below, indicate thatbirds inoculated with the above-described antigen composition orbacterin vaccine had a statistically lower rate of development ofintestinal lesions between Days 8 and 21. Lesion scores recorded on Day49 for both the control and vaccinated populations were notsignificantly different. Accordingly, it is believed that inoculationwith the antigen Composition of the invention induces an immune responseto the intestinal pathogen Clostridium perfringens thereby reducing theincidence, duration and/or severity of necrotic enteritis in avianpopulations.

Effect on Preharvest Intestinal Crop Bacteria.

All of the birds from the fourteen treatment groups described above wereadditionally inoculated with Escherichia coli (10⁶ per bird) andSalmonella spp. (10⁴ per bird) administered via oral gavage onpost-hatch Day 15. Eight birds (4 males and 4 females) from each controland each vaccine group were humanely euthanized and necropsied onpost-hatch Days 21 and 49. Crop content bacteria from each bird wereplated on an appropriate agar and fecal E. coli and Salmonella spp.bacteria counts were recorded.

The fecal bacteria data, summarized in TABLE 3, below, indicate thatbirds inoculated with above-described antigen composition or bacterinvaccine had a statistically lower concentration of both E. coli andSalmonella bacteria present in the crop contents. Accordingly, it isbelieved that inoculation with the antigen composition of the inventioninduces or stimulates an immune response to E. coli and Salmonellaresulting in reduced fecal bacteria content as well as reduced sheddingof bacteria.

Additionally, it was determined, as summarized in TABLE 3, below, thatbirds inoculated with the above-described antigen composition exhibitedan increase in average weight gain over the duration of the 49 daystudy.

TABLE 3 Day 21 Day 49 Criterion Control Vaccine Control Vaccine AverageLesion Score 1.018 0.250 0.179 0.107 Std Dev. 0.29 0.09 0.15 0.10 C.V.28.93 37.80 82.46 97.18 Fecal E. coli count (per ml) 2085.0 786.2 1479.9532.1 Std Dev. 158.80 138.90 161.02 67.59 C.V. 7.62 17.67 10.88 12.70Fecal Salmonella spp. 160.0 122.4 129.7 94.7 count (per ml) Std Dev.16.85 11.10 12.86 9.98 C.V. 10.53 9.07 9.92 10.54 Average Weight Gain(g) 547.648 580.171 2146.721 2225.416 Std Dev. 7.13 7.97 58.50 59.18C.V. 1.30 1.37 2.72 2.66

Effect on Marek's Disease Vaccine

A study was conducted to determine if the above-described antigencomposition or bacterin vaccine if administered in combination withcommercially available Marek's Disease vaccine negatively impacted thereplication of the vaccine viruses in cell culture or in vivo. Such anegative impact, as determined by decreases in the ability to re-isolatevaccine viruses at one week post-hatch, would suggest that the antigencomposition may decrease Marek's Disease vaccine efficacy.

Effect on Marek's Disease Vaccine in Culture.

To assess the effect of the above-described antigen composition onMarek's Disease vaccine preparations, the antigen composition and itsdiluent were obtained at 4× concentration. These were added to 4× stocksof HVT and SB-1 to generate 2× stocks of HVT and SB-1. Upon mixing ofequal amounts, this yielded 1× bivalent vaccines containing either 1×diluent or 1× antigen composition.

The vaccine stocks were titrated independently from the 4× stocks andalso titrated from each of the 1× final stocks. This was to determinethe effect of the antigen composition on HVT and SB-1 replication, inculture and to determine if the antigen composition would interfere withtitration of commercial vaccine. In each case, a commercial diluent wasused for diluting the vaccines. Vaccine, viruses and diluent wereobtained from commercial sources.

As indicated by the titration data, summarized in TABLE 4, below, andshown in FIG. 1, the antigen composition did not negatively affectMarek's Disease replication in cell culture. Titration of the vaccinestocks after either diluent or antigen composition addition showedessentially identical titers.

TABLE 4 Bird Dose Std Vaccine PFU/Vial Dose Dilution Mean Plaque # (PFU)Dev. HVT 1.59 × 10⁷ 4X 1:50 120.8 (±8.5) 6040 1028 SB-1  4.2 × 10⁵ 4X1:50   123 (±14) 3075 742 HVT + 1X 1:100 51.75 (±7.5) 5175 750 diluentSB-1 + 1X 1:100 20.75 (±6.4) 2075 640 diluent HVT + 1X 1:100  53.3(±3.9) 5325 386 antigen SB-1 + 1X 1:100  26.5 (±3.9) 2650 387 antigen

Effect on Marek's Disease Vaccine In Vivo

Eggs from a commercial broiler chicken strain, Ross X Cobb breed, wereinoculated at 18 days embryonic age with either a bivalent HVT/SB-1Marek's Disease vaccine (5000 PFU/bird HVT+2500 PFU/bird SB-1) mixedwith a control diluent (vaccine+diluent) or a vaccine including thebivalent Marek's Disease vaccine mixed with the above-described antigencomposition (vaccine+antigen). Post-hatch, an equal number of male andfemale chicks were randomly placed in grow out pens and grown underpractical commercial conditions.

At one week post-hatch chickens were bled via cardiac puncture,euthanized and the spleens were pooled into groups. The vaccine+diluentand vaccine+antigen groups were each comprised of four (4) pools ofthree (3) birds.

Blood and spleens were pooled and PBMC were purified from the wholeblood by histopaque centrifugation. Spleen cells were washed, countedand plated at 2×10 cells in triplicate dishes for each pool. PBMC werenot co-cultivated with CEF monolayers, as HVT and SB-1 infection ischaracteristically low at this time. At six (6) days post-plating, thedishes were examined and plaques for HVT and SB-1 were counted.

The above procedure was repeated three times over the course of four (4)weeks, i.e., a total of 16 pools of birds from the vaccine+diluent and atotal of 16 pools of birds from the vaccine+antigen groups wereinoculated and evaluated. The data obtained from the re-isolation countswere subjected to Chi-square and Students t-test analysis, the resultsof which are summarized in TABLE 5, below, and shown in FIG. 2.

TABLE 5 Vaccine + Diluent Vaccine + Antigen Group # Strain Count Group #Strain Count 1A HVT 48 ± 1 1B HVT 47 ± 13 SB-1 21 ± 3 SB-1 13 ± 1 2A HVT58 ± 14 2B HVT 56 ± 22 SB-1 16 ± 3 SB-1 19 ± 2 3A HVT 67 ± 16 3B HVT 37± 2 SB-1 17 ± 4 SB-1 15 ± 2 4A HVT 42 ± 1 4B HVT 69 ± 8 SB-1 25 ± 5 SB-120 ± 1 HVT Overall Average 54 HVT Overall Average 52 SB-1 OverallAverage 20 HVT Overall Average 17

The results in TABLE 5 indicate that comparable counts of HVT and SB-1plagues were obtained from the two treatment groups and, thus, overallno significant differences were found for either the HVT or the SB-1data.

In Week 4 of the study, a statistically significant difference was foundin the HVT counts between the vaccine+diluent and the vaccine+antigengroups. The antigen was found to increase the titers of HVT re-isolatedfrom inoculated chickens at one-week post-hatch. This is believed toindicate an advantage conferred on the replication of HVT. Conversely, asmall but statistically significant difference was found between SB-1re-isolated from the inoculated chickens.

Overall, the bacterin vaccine or antigen composition did not negativelyaffect Marek's Disease replication in vivo. Thus, it is unlikely thatthe antigen composition would decrease the efficacy of Marek's Diseasevaccines if employed in an in ovo vaccination program. Moreover, theaddition of the antigen composition should not negatively affect theability to titer vaccines.

While in the foregoing specification this invention has been describedin relation to certain embodiments thereof, and many details have beenput forth for the purpose of illustration, it will be apparent to thoseskilled in the art that the invention is susceptible to additionalembodiments and that certain of the details described herein can bevaried considerably without departing from the basic principles of theinvention.

1-20. (canceled)
 21. A multivalent antigen composition, comprising asuspension of dead or inactivated bacteria including: seven fieldstrains of E. coli consisting of Deposit Nos. ______, ______, ______,______, ______, ______, and ______, each field strain present in anapproximately equal amount; Pseudomonas aeruginosa (Deposit No. ______);Aerobacter aerogenes (Deposit No. ______); Salmonella enteritidis(Deposit No. ______); Salmonella typhimurium (Deposit No. ______);Salmonella agona (Deposit No. ______); and Salmonella Kentucky (DepositNo. ______), the multivalent antigen composition stimulating an immuneresponse in an avian species inoculated with said multivalent antigencomposition to at least one intestinal pathogenic organism selected fromthe group consisting of Clostridium perfringens, Salmonella species,Escherichia coli, and combinations thereof.
 22. The multivalent antigencomposition of claim 21, wherein the multivalent antigen composition isin ovo antigen vaccine.
 23. The multivalent antigen composition of claim21, further comprising a Marek's disease vaccine.
 24. The multivalentantigen composition of claim 23, wherein the suspension of dead orinactivated bacteria and the Marek's disease vaccine are present in aratio of 1:1.
 25. The multivalent antigen composition of claim 21,wherein the suspension of dead or inactivated bacteria is prepared by:separately culturing each bacteria at temperature of 35±1° C. for aperiod of 24±2 hours; sampling and plating each culture onto individualNutrient Agar plates; incubating the plated cultures for a period of24±2 hours at a temperature of about 35±1° C.; counting bacterialcolonies on the individual Nutrient Agar plates; mixing the individualbacteria cultures into an antigen composition in a select ratio based onthe colony counts; and autoclaving the antigen composition at 121° C.for 15±2 minutes.
 26. A bacterin vaccine, comprising: about 67% E. colistrains consisting of ATCC strain 25922 (Deposit No. ______), aUniversity of Delaware field isolate (Deposit No. ______), and fiveDelmarva field isolates (Deposit Nos. ______, ______, ______, ______,and ______), each strain of E. coli present in an approximately equalamount; about 10% Pseudomonas aeruginosa (Deposit No. ______); about 10%Aerobacter aerogenes (Deposit No. ______); about 4% Salmonellaenteritidis (Deposit No. ______); about 3% Salmonella typhimurium(Deposit No. ______); about 3% Salmonella agona (Deposit No. ______);and about 3% Salmonella Kentucky (Deposit No. ______), wherein thebacterin vaccine is prepared by: separately culturing each bacteria attemperature of 35±1° C. for a period of 24±2 hours; sampling and platingeach culture onto individual Nutrient Agar plates; incubating the platedcultures for a period of 24±2 hours at a temperature of about 35±1° C.;counting bacterial colonies on the individual Nutrient Agar plates;proportionately mixing the individual bacteria cultures into an antigencomposition based on the colony counts; and autoclaving the antigencomposition at 121° C. for 15±2 minutes.
 27. A method for reducingtransmission of pathogenic gastrointestinal organisms, comprising:inoculating an avian species in ovo at about 18 days embryonic age withthe vaccine according to claim
 26. 28. An in ovo vaccine, comprising: abacterin vaccine including: E. coli strains consisting of ATCC strain25922 (Deposit No. ______), a University of Delaware field isolate(Deposit No. ______), and five Delmarva field isolates (Deposit Nos.______, ______, ______, ______, and ______), and wherein each strain ofE. coli is present in substantially equal amounts, Pseudomonasaeruginosa (Deposit No. ______), Aerobacter aerogenes, (Deposit No.______) Salmonella enteritidis (Deposit No. ______), Salmonellatyphimurium (Deposit No. ______), Salmonella agona (Deposit No. ______),and Salmonella Kentucky (Deposit No. ______); and a Marek's diseasevaccine, the in ovo vaccine reducing a concentration of at least onepathogenic organism selected from the group consisting of E. coli spp.,Salmonella spp., or a combination thereof in a gastrointestinal tract ofan avian species inoculated in ovo with said vaccine.
 29. The in ovovaccine of claim 28, wherein the Marek's disease vaccine is selectedfrom the group consisting of HVT vaccines, SB-1 vaccines, andcombinations thereof.
 30. The in ovo vaccine of claim 28, wherein theavian species is a domestic fowl selected from the group consistingchickens, ducks, geese and turkeys.
 31. A method for reducingtransmission of pathogenic gastrointestinal organisms, comprising:inoculating an avian species in ovo at about 18 days embryonic age withthe vaccine according to claim
 28. 32. The in ovo vaccine of claim 28,wherein the bacterin vaccine is prepared by: separately culturing eachbacteria at temperature of 35±1° C. for a period of 24±2 hours; samplingand plating each culture onto individual Nutrient Agar plates;incubating the plated cultures for a period of 24±2 hours at atemperature of about 35±1° C.; counting bacterial colonies on theindividual Nutrient Agar plates; mixing the individual bacteria culturesinto an antigen composition in a select ratio of about 67:10:10:4:3:3:3:based on the colony counts; and autoclaving the antigen composition at121° C. for 15±2 minutes.
 33. The in ovo vaccine of claim 28, whereinthe bacterin vaccine and the Marek's disease vaccine are combined in aratio of from about 1:15 to about 15:1.
 34. The in ovo vaccine of claim33, wherein a dose comprises from about 0.005 ml to about 0.1 ml of thein ovo vaccine.